PROJECT SUMMARY The overall hypothesis to be tested in this proposal is that a novel class of nanocapsules can effectively deliver gene editing components into the two primary HIV-1 target cells, T-cells and macrophages, and mutagenize the HIV-1 provirus such that replication and/or reactivation from latency is aborted. While gene modification is challenging, the advantage over small molecule drugs is that the HIV-1 provirus or genes necessary for HIV-1 expression and/or infection can be directly knocked down or knocked out without the need to kill the infected cells. Efficient gene-modification activity has been achieved by a number of systems including zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), homing endonucleases, and most recently, the CRISPR/Cas9 system. Despite the promise of these new gene editing tools, therapeutic nucleic acids and proteins are rapidly lost from circulation and delivery vehicles cannot deliver gene modifying reagents by effective means to impact HIV-1 reservoirs. Thus, to date, all applications of gene modification for HIV-1 disease are currently practiced on cells removed from the body and transduced ex vivo. From our past experience with engineered lentiviral vectors, we recognize the difficult challenges of developing tools for in vivo gene editing, but also the promise and potential of bringing gene therapy into mainstream clinical practice. Our prior experience teaches us that viral vectors suffer from limitations in titer, adequate biodistribution, poor transduction of resting T-cells, complex genetic engineering, and immunogenicity. Recently, we developed a nanotechnology platform whereby individual macromolecules, protein, siRNA, gRNA, or DNA, are encapsulated and protected within a thin polymer shell by in situ polymerization of monomers and stabilized by environmentally responsive crosslinkers. In many respects, these ?nanocapsules? are similar to virion particles, being of similar size and, like virions, protect the single encased gene. However, they have the advantage of simple manufacturing to higher ?titer?, storage by freeze-dry, and, most importantly, the ability to easily alter the surface properties of chemical structure, charge, and ligand conjugation which determines factors such as biodistribution, cell binding, and entry. Since the properties of the nanocapsule are conferred by the shell which shields the cargo, virtually any nucleic acid or protein cargo can be interchanged. By judicious choice of polymer shell and crosslinkers, we successfully engineered nanocapsules which enhance biodistribution to reservoir sites, release a model cargo in time release fashion, and target specific cells in vivo through ligand recognition of cell surface molecules. Furthermore, these nanocapsules themselves are relatively non- immunogenic and shield the cargo from the immune system. These proof of principle studies begin to overcome the challenges outlined above and thus provide the basis for our proposed studies.